Anion-Sensitive Fluorophore Identifies the Drosophila ...Anion-Sensitive Fluorophore Identifies the...

Anion-Sensitive Fluorophore Identifies the DrosophilaSwell-Activated Chloride Channel in a Genome-WideRNA Interference ScreenStephanie C. Stotz1, David E. Clapham1,2,3*

1 Howard Hughes Medical Institute, Department of Cardiology, Boston Children’s Hospital, Boston, Massachusetts, United States of America, 2 Manton Center for Orphan

Disease, Boston Children’s Hospital, Boston, Massachusetts, United States of America, 3 Department of Neurobiology, Harvard Medical School, Boston, Massachusetts,

United States of America

Abstract

When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Clswell) activate to reduce intracellular osmolalityand prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Clswell

channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stablyexpressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila Clswell channel. Ofthe 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targetingdBest1 eliminated the Clswell current (IClswell). We further demonstrate the essential contribution of dBest1 to DrosophilaIClswell with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94Cconstruct in Drosophila cells significantly reduced the endogenous IClswell. We confirm that exogenous expression of dBest1alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila–like IClswell. In contrast, activationof mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues ofdBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, stronglyindicating that the Bestrophin protein forms the Clswell channel itself rather than functioning as an essential auxiliarysubunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.

Citation: Stotz SC, Clapham DE (2012) Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNAInterference Screen. PLoS ONE 7(10): e46865. doi:10.1371/journal.pone.0046865

Editor: Alexander A. Mongin, Albany Medical College, United States of America

Received June 12, 2012; Accepted September 6, 2012; Published October 4, 2012

Copyright: � 2012 Stotz, Clapham. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: SCS is supported by the Tommy Kaplan Fellowship, Boston Children’s Hospital, Boston. Howard Hughes Medical Institute provided funding for thisstudy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

All mammalian cells express chloride channels activated by

decreases in extracellular osmolality, albeit with different biophys-

ical properties [1]. The ubiquitous expression of Clswell suggests its

essential cellular function. Tightly regulated Clswell channels

participate in volume regulation, motility, cell survival, and cell

division [1]. In contrast, de-regulated constitutively active Clswell

channels exacerbate several cardiac diseases, including myocardial

hypertrophy and heart failure [2]. The mammalian Clswell

channel- encoding gene has yet to be identified despite the wealth

of proteins nominated by candidate approaches [3]. These

proteins include ClC-2 [4], ClC-3 [5], P-glycoprotein [6,7], pICln

[8,9], p64 [10], phospholemman [11], Best1 and 2 [12],

TMEM16A [13], and TMEM16F [14]. The research community

has yet to agree on any of these candidates as a bona fide Clswell

channel.

In Drosophila, however, accumulating evidence indicates that

dBest1 encodes for a Clswell channel. RNAi targeting dBest1

eliminates Drosophila Schneider (S2) cell IClswell, an effect rescued

by re-introduction of dBest1 [15]. Further, swell activated dBest1

mutants have altered biophysical properties and reactivity to

sulfhydryl reagents [16]. dBest1 likely forms the chloride

conducting pore, but it may be an obligate auxillary subunit of

Drosophila IClswell that modifies channel properties akin to CaV bsubunits [17].

Assigning chloride channel function to any protein is difficult.

The known chloride channel families (e.g., ClC, Anoctamin/

TMEM16, CFTR, and ionotropic GABAA and GlyR) lack

structural pore or gating motifs that might form the basis for in

silico identification. Expression cloning approaches have also failed

due to widespread Clswell channel expression that precludes the

separation of endogenous and over-expressed protein activities.

Moreover, known chloride channels blockers are non-specific and

their affinities are far too low to encourage affinity purification.

Finally, previous chloride indicators are poor tools for screening

due to loading and retention issues, inconsistent results, and poor

reproducibility [18].

Here we present an unbiased genome-wide, high-throughput

RNAi screen designed to identify the Drosophila Clswell channel and

its regulators. Our screen employed H148Q-YFP, a genetically

encoded anion-sensitive yellow fluorescent protein [19], to report

Clswell activity in Drosophila S2R+ cells. Of our 595 initial hits that

altered chloride handling, we concentrated on characterizing

proteins with mammalian homology and at least one transmem-

brane domain as potential Clswell channels. dBest1 emerged from

our screen as the lead candidate for Drosophila Clswell. Both RNAi

PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46865

knockdown of dBest1 and overexpression of a dominant-negative

dBest1 eliminated the Clswell current in Drosophila S2R+ cells.

Conversely, dBest1 overexpression in a mammalian system (HEK

cells) produced a Drosophila–like IClswell. To identify domains

necessary for swell activation we characterized chimeras between

the swell-sensitive dBest1 and the swell-insensitive mBest2. Swell

sensitivity is only apparent in mBest2, the closest mammalian

homolog of dBest1, when the protein contains the dBest1 amino

(N)-terminus. This chimera maintains the pore properties of

mBest2, providing additional evidence that the protein itself forms

a channel rather than functioning as a necessary auxiliary subunit.

We conclude that dBest1 is the channel underlying the Drosophila

IClswell.

Results

Drosophila S2R+ Cells have Robust IClswell

Drosophila S2 cells are used extensively in genome-wide RNAi

screens to dissect signaling pathways, determine protein functions,

and assign protein molecular identity [20]. These macrophage-like

cells, derived from primary culture of late stage Drosophila

melanogaster embryos [21] readily take up RNAi from serum-free

media. The subsequent process of targeted mRNA ablation is

efficient and highly reproducible [22,23]. For our screen we used

S2R+ cells [24], an adherent S2 variant well suited for assays that

require multiple solution changes. Importantly, S2R+ cells have a

consistent, large IClswell that activates slowly upon a drop in

extracellular osmolality (Figure 1A). IClswell starts to activate within

2 min exposure to hypo-osmotic media, reaching steady state

activation by 5 min. The fully activated Clswell conductance is

anion selective (Figure 1B). The relative permeability sequence of

S2R+ Clswell is I = SCN.Cl.MES.aspartate (ASP) while the

slope conductance sequence is I = SCN = Cl.MES.ASP (Table 1

& 2). The Clswell I–V relationship has a slight ‘‘S’’ shape, revealing

rectification. An extended step protocol further illustrates features

of S2R+ Clswell (Figure 1C). IClswell exhibits an initial instantaneous

activation followed by a second slow activation phase, suggesting

that more than one type of Cl2 conductance is turned on in S2R+cells with cell swelling. In contrast, Chien & Hartzell [15] reported

a single phase, time-independent IClswell activation in their S2 cells,

perhaps indicating Cl2 channel expression differences in the two

cell lines. Tail currents, normally indicating time dependence of

deactivation of the channel, are evident in our recordings.

However, since tail currents were not observed under symmetrical

recording conditions [15], we attribute these currents to the exit of

intracellular Cl2 accumulated during the prolonged steps. S2R+IClswell has an interesting pharmacological profile (Figure 2A).

Even at a high concentration (100 mM), the non-specific chloride

channel blocker 4,49-diisothiocyano-2,29-stilbenedisulfonic acid

(DIDS) [25] blocks less than 25% of the S2R+ IClswell

(Figure 2A). 4-2(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-

yl (DCPIB), a mammalian selective IClswell blocker [26], fails to

completely block S2R+ IClswell at 30 mM (Figure 2A & E). DCPIB

blocks in a voltage-dependent manner (Figure 2E); at 0 mV 52%of S2R+ IClswell is blocked, while 90% is blocked at 80 mV.

Surprisingly, furosemide, a Na-K-2Cl cotransporter (NKCC;

SLC12A2) blocker, almost completely inhibits S2R+ IClswell at

1 mM (Figure 2A & 3C).

H148Q-YFP Reliably Reports the Activity of S2R+ CellClswell Channels

Clswell conducts iodide better than chloride, favoring the use of

the H148Q-YFP indicator as a reporter of its activity (I2

KD = 20 mM and Cl2 KD = 100 mM [19,27,28]). Several anion-

sensitive YFP variants accurately quantify intracellular Cl2

concentration or changes [27,29–31]; anion binding near the

YFP chromophore suppresses fluorescence emission by altering

chromophore resonance [19]. H148Q-YFP was chosen for Clswell

detection because it is bright and potently suppressed by I2; these

properties are critical for good signal-to-noise ratios during

screening. H148Q-YFP (pKa = 6.7) is also sensitive to intracellular

pH changes [19,28]. S2R+ cells stably expressing H148Q-YFP

maintain their fluorescence in 240 mOSM NaCl however

(Figure 3A), indicating that cell swelling does not appreciably

alter intracellular pH. Subsequent replacement of bath Cl2 with

I2 rapidly suppresses indicator fluorescence by 50% as I2 enters

the cells through open channels and interacts with the probe. The

large fluorescence change and low intrinsic assay variability favor

clear separation of potential hits. In the absence of hypo-osmotic

solution, I2 is unable to enter the S2R+ cells and fluorescence is

maintained (Figure 3B), indicating that S2R+ cells lack alternative

constitutively active I2 entry pathways that could confound our

ability to identify the Clswell channel. Further, furosemide block of

open Clswell channels prevents appreciable fluorescence suppres-

sion (Figure 3C & D), suggesting that RNAi effectively targeting

the Clswell channel will be readily identifiable as hits.

Genome-wide RNAi Screening of H148Q-YFP S2R+ CellsIdentifies dBest1 as the Clswell Channel

The primary screen was conducted at the Harvard/HHMI

Drosophila RNAi Screening Center using our stable H148Q-YFP-

expressing S2R+ cell line. Each well of sixty-six 384-well assay

plates contained a dsRNA targeting 1 of 13,900 genes encoding

proteins or non-coding RNAs (DRSC 2.0; Figure 4A). Five days

after S2R+ cells were treated with RNAi, we assessed cellular

fluorescence under swell conditions in the presence of Cl2 and I2.

Wells with fluorescence or ratio (I2 fluorescence/Cl2 fluorescence)

changes greater than 1.5 times the standard deviation of the plate

mean were initially considered as hits (Clswell channel candidates

or regulators of its activation pathway). We pared the list of 595

hits to genes with mammalian homologs and those with predicted

transmembrane domains (Figure 4B, Table S1). In a secondary

screen, we confirmed that each RNAi significantly reduced

swelling-induced fluorescence and targeted only the mRNA from

the identified gene (qPCR). We then directly measured IClswell via

whole-cell voltage clamp. Candidates genes, whose RNAi signif-

icantly reduced the S2R+ cell IClswell, were cloned and expressed in

HEK293 or CHO-K1 cells. IClswell was then measured via whole-

cell recording and compared with currents from untransfected

cells. The only candidate of our screen to satisfy all the criteria for

a Clswell channel was dBest1 (Table S1).

DRSC26457 RNAi Targeting dBest1 Eliminates IClswell

dBest1 is a protein of 769 amino acids containing 4

transmembrane domains [32,33] (Figure 4C). It is one of four

Bestrophin family members in Drosophila, with highest homology to

mBest2/hBest2 (51% identity and 67% similarity; BLAST).

Hartzell and colleagues first proposed that dBest1 was a chloride

channel activated by high intracellular Ca2+ and cell swelling

[15,16]. In our H148Q-YFP fluorescence assay dBest1 RNAi

DRSC26457 abrogated the fluorescence change normally ob-

served when I2 enters the S2R+ cells through activated Clswell

conductances (Figure 5A, B). Interestingly, DRSC26457 also

decreased the baseline fluorescence variability of S2R+ cells

(Figure 5B), suggesting that IdBest1 contributes to resting intracel-

lular Cl2 concentrations. S2R+ IClswell was essentially eliminated

by dBest1 RNAi DRSC26457 treatment (Figure 5C). This RNAi

specifically and effectively reduced dBest1 mRNA by 91.5% 60.5

Identifying the Swell Activated Chloride Channel


(n = 3; qPCR); mRNA levels for the 3 remaining Bestrophin

members and other Clswell candidates were unaffected. A second

RNAi targeting dBest1 (DRSC16909; corresponds with dB1S

[15]) was part of our initial screen. It was less effective at knocking

down dBest1 mRNA (85% reduction, n = 3; significantly less than

DRSC26457; p,0.001, Student’s t-test) and had two predicted

off-target hits: CG4623 (20/20) and CG16711 (18/18).

DRSC16909 did not significantly alter H148Q-YFP I–induced

Figure 1. Characterization of the S2R+ cell IClswell. (A) Hypo-osmotic solutions slowly activate IClswell. IClswell begins to activate 1.760.3 min afterexposure to 200 mOSM solution and reaches steady state activation within 560.3 min (n = 13). IClswell was assessed in ramp protocols and reported at+84.5 mV (upper trace) and -115.5 mV (lower trace). 240 mOSM stimulates IClswell activation slightly more slowly (1.860.3 min to initiation and5.260.6 min to steady state, n = 12; data not shown). (B) The S2R+ cell IClswell is anion-selective. IClswell was activated by 200 mOSM solution; relativepermeability and slope conductance sequences were determined for the steady state IClswell by replacing Cl2 with equimolar anion concentrations.(C) An extended step protocol (red inset) reveals more than one set of activation kinetics with offset activation initiation times.doi:10.1371/journal.pone.0046865.g001



Figure 2. Pharmacological profiles of S2R+ IClswell and IdBest1 match and differ from those of HEK IClswell and Id64m. (A–D) % Block ofS2R+ IClswell, IdBest1, HEK IClswell and Id64m by 1 mM furosemide, 100 mM DIDS, and 30 mM DCPIB. Block at 0 mV is presented to emphasize theincomplete voltage-dependent DCPIB block of S2R+ IClswell and IdBest1. (A) Steady state S2R+ IClswell activated by 200 mOSM stimulation was blocked96% 61.6 by furosemide, 19% 64 by DIDS, and 52% 610.6 by DCPIB. * no difference compared to IdBest1 block and significantly different comparedto HEK IClswell and Id64m (ANOVA, p,0.05). (B) IdBest1, stimulated for 2 min by 200 mOSM, was blocked 96% 61.7 by furosemide, 44% 610 by DIDS,and 47% 610.9 by DCPIB. * no difference compared to S2R+ IClswell block and significantly different compared to HEK IClswell and Id64m (ANOVA,p,0.05). (C) Steady-state HEK IClswell activated by 200 mOSM stimulation was blocked 7% 63.5 by furosemide, 77% 63 by DIDS, and 99% 60.7 byDCPIB. (D) Constitutive Id64m (320 mOSM) was blocked 77% 63 by furosemide, 98% 61.5 by DIDS, and 98% 61.2 by DCPIB. � significantly differentcompared to S2R+ IClswell, HEK IClswell and Id64m (ANOVA, p,0.05). (E–F) I–V relations for S2R+ IClswell and IdBest1 demonstrate DCPIB voltage-dependentblock. At 80 mV, DCPIB block of S2R+ IClswell is 90% 63.6 (n = 6), and 82% 66.5 for IdBest1 (n = 7).doi:10.1371/journal.pone.0046865.g002



fluorescence suppression (Figure 5A), and was not a hit in our

initial screen. It is possible that the 15% remaining mRNA

translated sufficient amounts of functional dBest1/Clswell channels

to exclude it as a hit in our screen. This prospect emphasizes the

importance of validated, effective RNAi for accurate screening.

Mutant dBest1 W94C Significantly Reduces S2R+ IClswell

To substantiate the conclusion that dBest1 is an essential

component of the Clswell channel, we tested whether a mutant

dBest1 would act as a dominant negative regulator of IClswell. In

humans, Bestrophin 1 is mutant in vitelliform macular dystrophy

Figure 3. H148Q-YFP stably expressed in S2R+ cells reports the entry of I2 through activated Clswell channels. (A) Cellular swelling in240 mOSM Cl2 did not alter fluorescence intensity as Clswell channels activate (Student’s t-test, p = 0.65; n = 76). Replacement of Cl2 with I2 evoked a51% 61.3 decrease in fluorescence (Student’s t-test, p,0.001; n = 76). Imaging assay; fluorescence is in arbitrary units (a.u.). (B) Clswell channels mustbe open for I–induced fluorescence suppression to occur. 320 mOSM NaI suppresses fluorescence by 16% 60.7 (Student’s t-test, p = 0.1; n = 54). (C)Furosemide, an NKCC2 blocker, completely inhibits the S2R+ cell Clswell channels at 1 mM (n = 3). (D) 1 mM Furosemide block of Clswell preventssignificant I–induced suppression of H148Q-YFP fluorescence (Student’s t-test, p = 0.54; n = 16).doi:10.1371/journal.pone.0046865.g003

Table 1. Relative Permeabilities.

PX/PCl n

I SCN Cl MES ASP

S2R+ 1.560.12 1.660.15 1 0.7460.15 0.0760.015 3

dBest1 1.860.05 260.04 1 0.6360.04 0.0960.0017 5

mBest2 1.760.15 2.960.68 1 0.4560.14 0.0560.006 6

d64m 2.260.34 2.660.11 1 0.4360.14 0.0760.011 4

HEK 1.560.06 1.860.2 1 0.6260.005 0.1160.003 6

doi:10.1371/journal.pone.0046865.t001

Table 2. Slope Conductance.

Slope Conductance (G; I/V) n

I SCN Cl MES ASP

S2R+ 29.666.8 26.965.9 2867.5 16.463.5 7.661.5 3

dBest1 8.261.5 6.661.1 5.260.7 3.360.4 1.960.3 5

mBest2 1064.9 2.460.95 6.663.4 261.3 1.761.1 6

d64m 1063.5 2.560.36 4.460.3 1.960.5 1.760.3 3

HEK 26.268.2 24.967.1 26.967.9 9.565.4 5.462.7 6

doi:10.1371/journal.pone.0046865.t002



Figure 4. Genome-wide RNAi screening of H148Q-YFP S2R+ cells identifies Clswell channel candidates and regulators. (A) RNAitreatment alters S2R+ cell H148Q-YFP fluorescence levels. Heat map plate reader data following 5d RNAi treatment (240 mOSM Cl2). Fluorescencewas subsequently measured in 240 I2. Wells where the I2 to Cl2fluorescence ratio was high are hits. Control RNAis are found in columns 13 and 14.Thread RNAi is in 13B, 13G, 14K, and 14N. Rho RNAi is in 13D, 13E, 14J, and 14O. GFP RNAi is in 13C, 13F, 14L, and 14M. Wells with elevatedfluorescence in 240 mOSM Cl2 are shown in red and orange. (B) Functional classification of the 595 hits identified in our screen. 21 hits weretransmembrane proteins of unknown function, putative ion channels, or transporters. 9 candidates with human homology were further evaluated(Table S1). (C) Protein sequence alignment of dBest1 and mBest2 (Multalign; multalin.toulouse.inra.fr/multalin/). Green bars indicate transmembranedomains. Yellow bars indicate other putative a-helices (SOSUI; bp.nuap.nagoya-u.ac.jp/sosui/). A red star indicates the W94C mutation. A blue starindicates the Ca2+-binding bowl. A pink arrow indicates Stop 383. A pink box outlines the region switched in the d64m chimera.doi:10.1371/journal.pone.0046865.g004

Figure 5. DRSC26457 identifies dBest1 as Clswell. (A & B) RNAi efficiently targeting dBest1 prevents significant I–induced H148Q-YFPsuppression following hypo-osmotic stimulation. Fluorescence is in arbitrary units (a.u.). (A) Plate reader assay. DRSC26457 RNAi treatment resulted ina fluorescence decrease of 6.4% 619. Control and DRSC16909 RNAi treatment resulted in fluorescence decreases of 40.5% 69.1 and 43.7% 66.2respectively. * 240 mOSM NaCl and NaI fluorescence levels are significantly different (Student’s t-test, p,0.05). (B) Imaging assay. The fluorescencelevels of individual S2R+ cells treated with control or dBest1 DRSC26457 RNAi were measured during hypo-osmotic stimulation in the sequentialpresence of Cl2 and I2. The fluorescence of control cells decreased by 56% (n = 44); in contrast, the fluorescence of dBest1 DRSC26457 RNAi treatedcells was suppressed by 15% (n = 174). (C) dBest1 DRSC26457 RNAi eliminated IClswell in S2R+ cells. Following dBest1 RNAi treatment I320 is notsignificantly different from I240 (Student’s t-test, p = 0.1). * control and RNAi treated I240 are significantly different (Student’s t-test, p = 0.02). (D) dBest1W94C-gfp overexpression suppresses S2R+ IClswell. * control and W94C-gfp I200 mOSM are significantly different (Student’s t-test, p = 0.02). (E) Confocalimages of dBest1 W94C-gfp overexpression in S2R+ cells. Images were obtained before (320 mOSM) and after swell (200 mOSM). Scale bar indicates10 mm.doi:10.1371/journal.pone.0046865.g005



(Best’s disease [34,35]). One mutation, W93C, occurs in a

conserved sequence of the channel’s putative pore [36,37]

(Figure 4C). When we expressed the homolog dBest1 W94C-gfp

in S2R+ cells, IClswell was significantly reduced (Figure 5D).

Interestingly, the late activating component of IClswell remained

clearly evident at depolarized potentials (Figure S1). We could not

study this current in more detail as a loss of cell membrane

integrity rapidly ensued. We conclude that dBest1 is responsible

for the early activating S2R+ cell IClswell. W94C might interact

with WT dBest1 to disrupt the Clswell channel pore or it may

prevent proper protein trafficking [38]. In S2R+ cells, dBest1

W94C-gfp has a distinct intracellular expression pattern unaltered

by osmotic changes (Figure 5E), suggesting the latter explanation

over the former. Regardless, dBest1 W94C has a dominant

negative impact on IClswell, further evidence that dBest1 is integral

to the Clswell channel.

Another disease-associated Bestrophin mutation, D308A, occurs

in a putative Ca2+ -binding bowl located in the channel’s C-

terminus (Figure 4C; blue star). D308A is proposed to eliminate

Bestrophin activation by disruption of calcium binding [39]. We

introduced this mutation into dBest1 to determine if activation by

calcium and cell swelling could be separated. Unfortunately

dBest1 D308A-gfp was not functional in HEK cells (data not

shown). Three possible explanations may underlie this result: 1)

activation by multi-modal stimuli is simultaneously disrupted by

the mutation; 2) the mutation causes protein misfolding and the

channel function has been eliminated for reasons unrelated to

activation; 3) the mutant channel is mislocalized. Our GFP-tagged

protein was expressed (data not shown), but we cannot exclude the

possibility that it mislocalizes or fails to interact appropriately with

other proteins necessary for IClswell activation or channel function

[34].

Exogenous dBest1 Expression Creates a Drosophila-likeIClswell

Exogenous expression of a candidate protein substantiates

whether the protein is necessary and/or sufficient in a given

process. Our secondary screen assessed whether candidate protein

expression resulted in a novel IClswell or augmented the endoge-

nous HEK IClswell (Table S1). The HEK cell line chosen for

candidate over-expression lacked constitutive ICl and ISCN

(potentially contaminating conductances attributable to SLC1A

family member expression [40]; data not shown). The endogenous

HEK IClswell develops very slowly (Figure 6A & B); a two fold

increase was noted within the first 2 min of swell. Once the HEK

IClswell reaches steady state, however, it has increased more than

forty fold (44.4610.7 fold, n = 29; Student’s t-test, p,0.000005).

Tail currents are absent (Figure 6B & C). Characteristic voltage-

dependent inactivation develops during steps to positive potentials

(Figure 6C). HEK IClswell is anion selective; its permeability and

conductance sequences match closely to those of S2R+ IClswell

(Figure 6D; Table 1 & 2). The HEK IClswell pharmacological

profile (Figure 2C) correlates well with the literature. 100 mM

DIDS, slightly above the reported IC50 [41], blocks 78% of the

HEK IClswell at +80 mV (Figure 2C). DCPIB has an IC50 of 4 mM

[26]; at 30 mM 100% of HEK IClswell is blocked (Figure 2C).

1 mM furosemide barely inhibits HEK IClswell (Figure 2C). The

endogenous HEK IClswell recapitulates the key features noted for

the mammalian IClswell [1].

Bestrophin proteins are not universally accepted as bona fide

chloride channels; alternatively they are intracellular ion channel

regulators [33,42,43]. dBest1-gfp is clearly observed on or near the

surface of HEK-293 cells (Figure 7A). Its expression results in a

Drosophila-like IClswell (Figure 7B–E). Constitutively active IdBest1 is

apparent in iso-osmotic 320 mOSM solution and is significantly

increased 1664.5 fold (Student’s t-test, p,0.005) during the first

2 min of hypo-osmotic stimulation (Figure 7B). IdBest1 has the same

‘‘S’’-shaped rectification as Drosophila IClswell during ramps

(Figure 7C); tail currents and time-dependent activation are both

apparent in the step protocol (Figure 7d). IdBest1 is anion selective;

it has the same permeability and conductance sequences as S2R+IClswell and HEK IClswell (Figure 7E; Table 1 & 2). Strikingly, IdBest1

and the endogenous S2R+ IClswell share a similar pharmacological

profile that differs significantly from HEK IClswell (Figure 2A–C).

100 mM DIDS inhibits 35% of IdBest, while 30 mM DCPIB blocks

45%. 1 mM furosemide blocks nearly 100% of the IdBest1. We

conclude that dBest1 expression results in a Drosophila-like IClswell;

it cannot be attributed to endogenous HEK IClswell upregulation.

dBest1 Swell Activation can be Conferred on the Swell-insensitive mBest2

The structural domains necessary for swell-induced channel

activation are unknown. Although dBest1 has a long poorly

conserved C-terminus (Figure 4C), it is not necessary for swell

activation. dBest1 remains swell-sensitive despite the removal of up

to 338 of its C-terminal amino acid residues (Stop 383, Figure 4C;

Figure 8A). Next we examined whether chimeras might reveal the

domains underlying swell activation. dBest1’s closest mammalian

homolog, mBest2, is not activated by hypo-osmotic solutions

(Figure 8B). Chimera d64m (the first 64 residues are dBest1; the

remaining residues are identical to those of mBest2; Figure 4C)

expression resulted in a constitutively active current that more

than doubled with swelling (2.3 fold 60.3 increase; Student’s t-test,

p,0.05; Figure 8B–D). The d64m chimera maintained the

relative permeability and slope conductance of mBest2 (Figure 8E

& F; Table 1 & 2), suggesting that the channel’s pore domain is

downstream of residue 64. Two other groups have assessed

mBest2 selectivity [44,45] and found greater permeability for SCN

than we report here. Both groups used high intracellular calcium

to activate ImBest2; we report constitutive ImBest2 measured with

high internal calcium buffering (i.e. ,10 nM free calcium). Our

HEK cell line was also screened for potentially contaminating ISCN

(data not shown) attributable to SLC1A family member expression

[40]. The pharmacological profile of Id64m noticeably diverged

from both that of HEK IClswell and IdBest1 (Figure 2). Furosemide

blocked 75% of Id64m, while DIDS and DCPIB both blocked Id64m

to near completion (Figure 2D). We conclude that the dBest1 N-

terminal domain is required for swell activation of the mBest2

channel. The reverse chimera (m64d) was nonfunctional; exoge-

nous currents were not observed with swelling or in the presence of

high intracellular Ca2+ (data not shown). We cannot conclude with

this data however, that the N-terminus is a ‘‘swelling’’ domain as it

lacks any predictive motifs. We hypothesize that it works in

concert with domains present both in dBest1 and mBest2 to

facilitate swell activation. The strong correlation between S2R+IClswell and IdBest1, combined with the unique selectivity and

pharmacology of the d64m chimera, support the conclusion that

the Bestrophin protein itself forms the Clswell channel rather than

functioning as an auxiliary subunit.

Discussion

Our study validates the H148Q-YFP fluorophore as a reliable

reporter of Clswell channel activity in genome-wide RNAi

screening studies. H148Q-YFP has been employed very effectively

in the identification of novel chloride channel activators,

modulators, and blockers of CFTR and Ca2+-activated Cl2

channels [18]. This is the first reported RNAi screen using an



Figure 6. The endogenous HEK cell IClswell has characteristic mammalian IClswell properties. (A) HEK cell IClswell develops slowly, reachingsteady state after 5 min exposure to 200 mOSM solution. X indicate min in 200 mOSM. (B) Ramp protocol (inset) assessment of the HEK IClswell. LittleIClswell has developed after 2 min in 200 mOSM solution. The steady state IClswell is outwardly rectifying and inactivating at positive potentials. No tailcurrents are apparent. (C) Step protocol (inset) assessment of the HEK IClswell. Rapid inactivation is observed at positive potentials. No tail currents areapparent. (D) Relative permeability and slope conductance sequences for the endogenous HEK IClswell are SCN = I.Cl..MES.ASP vsSCN = I = Cl..MES.ASP.doi:10.1371/journal.pone.0046865.g006



anion-sensitive fluorescent protein to assign molecular identity to a

chloride channel. Our screen supports the findings of the Hartzell

lab [15]: dBest1 RNAi eliminates Drosophila IClswell.

We found that the RNAi effectiveness was essential for Clswell

candidate identification. Two separate dBest1-targeting RNAi’s

were part of our initial screen: DRSC26457 and DRSC16909

Figure 7. dBest1 overexpression in HEK cells produces a S2R+ cell-like IClswell. (A) dBest1-gfp targets to the membrane of HEK cells.Confocal images of dBest1-gfp overexpressed in HEK-293 cells. The DIC image is on the left, GFP in the middle, overlapped images on the right. Scalebar indicates 10 mm. (B) IdBest1 rapidly develops within the first 2 min of hypo-osmotic stimulation. (C) The developing IdBest1 has the same ‘‘S’’ shaperectification as the endogenous S2R+ cell IClswell. (D) Step protocol shows that IdBest1 shares time-dependent activation and tail current propertieswith S2R+ cell IClswell. (E) The constitutively active IdBest1 and S2R+ cell IClswell selectivity sequences are very similar. (F) IdBest1 is clearly separable fromthe endogenous HEK cell IClswell. IdBest1 increases 15.8 fold 64.5 (n = 18; ** paired Student’s t-test, p,0.005) in the first 2 min of hypo-osmoticstimulation; the endogenous HEK cell IClswell increases 2.1 fold 60.4 (n = 29; * paired Student’s t-test, p,0.05).doi:10.1371/journal.pone.0046865.g007



(which corresponds to dB1S [15]), but only DRSC26457 was a

hit. qPCR reported a 95% reduction in dBest1 mRNA with

DRSC26457 treatment versus an 85% reduction with

DRSC16909. Hartzell and colleagues found that S2 cell IClswell

was significantly reduced following treatment with 0.4 mg of

DRSC16909 [15], while in our screen each assay well had a

standardized 0.25 mg of RNAi. Using more RNAi may have

effected greater target knockdown and resulted in the detection

of DRSC16909 as a hit in our screen. This result emphasizes

the importance of RNAi effectiveness in hit identification.

Exogenously expressed IdBest1 and endogenous S2R+ IClswell

share similar characteristics, including time-dependent activation,

tail currents, relative permeability sequences, slope conductance

sequences, and pharmacological profiles. The shared properties of

Drosophila IClswell and IdBest1 suggest that the same protein forms the

channel responsible for both. Bestrophin is a known ion channel

modulator, altering voltage-gated calcium channel activity [46]. If

Figure 8. Chimeras between dBest1 and mBest2 confer swell activation on mBest2. (A) Truncation of dBest1 does not interfere with swellsensitivity. Constitutive currents are apparent with both dBest1 and Stop 383 overexpression in iso-osmotic solutions (320 mOSM). With swell, currentincreased dramatically for both constructs (Student’s t-test, * p,0.05, ** p,0.005). (B) d94m current increases significantly within the first 2 min ofswelling (* Student’s t-test, p,0.05). (C) Time course for d64m swell activation. (D) Current-voltage relations for constitutive d64m, and following2 min 200 mOSM solution. (E) Selectivity sequence for the constitutively active d64m current. (F) Selectivity sequence for the constitutively activemBest2 currents.doi:10.1371/journal.pone.0046865.g008



dBest1 expression simply modulated or upregulated the endoge-

nous HEK Clswell channel expression, we would have expected the

resulting IClswell to maintain the properties of HEK IClswell. Instead

we observed that dBest1 introduced an exogenous Drosophila-like

IClswell whose development preceeded that of the endogenous

HEK IClswell. IdBest1 matched the pharmacological profile of the

S2R+ IClswell. Moreover, we found that the exogenous Drosophila-

like IClswell permeability and conductance sequence could be

transformed into that of mBest2 with the d64m chimera. The

pharmacological profile of Id64m was again significantly different

from the endogenous HEK IClswell. We conclude that dBest1 is the

Drosophila Clswell channel.

Several Bestrophin mutations are associated with vitelliform

macular dystrophy [36,37]. How these mutations are causally

linked to the disease is not clear. Here we found that

overexpression of the disease-linked W94C dBest1 mutant in

S2R+ cells significantly suppressed the endogenous Drosophila

IClswell. The W94C mutation occurs in the putative pore of dBest1

and thus may disrupt Clswell conductance. However, the fluores-

cently tagged W94C dBest1 protein appears to localize to

intracellular compartments, consistent with mislocalization. Mile-

nkovic et al, have recently proposed that disease-associated

Bestrophin mutations cause defects in intracellular trafficking

[38]. Both scenarios may explain the dominant negative effect of

dBest1 W94C on Drosophila IClswell: non-functional, pore-disrupt-

ing, mutant Bestrophin proteins complexing with wild-type dBest1

may be largely retained within the endoplasmic reticulum. The

end result would be the elimination of endogenous Drosophila

IClswell. Our experiments support the hypothesis that mutant

Bestrophin W93C expression could significantly disrupt chloride

flux and homeostasis in the human macula, contributing to the

disease state.

The distinction of Bestrophin function in Drosophila versus

mammalian cells is most clearly illustrated by Hartzell and

colleagues [16]. IClswell measured in peritoneal mast cells isolated

from mBest12/2, mBest22/2, and mBest1/2 double knockout mice

was identical to wild-type IClswell. hBest1 and mBest2 are swell

sensitive in that their currents are inhibited by hyperosmotic

solutions. However, their activity does not increase with swell [12].

We confirm here that mBest2 activation does not increase when

cells swell. Our d64m chimera contained only a small portion of

dBest1, yet it responded to cellular swelling. The crucial N-

terminal region contains no distinct association domains or

predictive structures that might explain its coupling to changes

in cell stretch, tension, or osmolality. We speculate that the N-

terminus contributes to a required tertiary structure that enables

swell signaling events to activate the dBest1 channel.

Our genome-wide RNAi screen of S2R+ cells and follow-up

study firmly establishes that the dBest1 protein forms the Drosophila

Clswell channel. It further validates a live cell genetically

engineered fluorescent screening platform to identify other

mammalian chloride channels.

Materials and Methods

Generation of the S2R+ YFP- H148Q Stable Cell LineThe stable S2R+ cell line expressing a halide-sensitive YFP

(H148Q-YFP; kindly provided by Dr. Alan Verkman, UCSF) was

generated with a selection vector (pCoBlast). H148Q-YFP was

subcloned into the pAc5.1/V5-HisA vector (Invitrogen, CA). The

S2R+ cells were transfected by electroporation (Amaxa cell line

nucleofector kit V; Lonza). Cells were placed under selective

pressure with 25 mg/ml blasticidin for 2 weeks. Two rounds of

fluorescence-activated cell sorting (FACS; DFCI Flow Cytometry

Core Facility) normalized YFP fluorescence intensities. These

S2R+ cells exhibited a robust IClswell as described in Figure 1.

S2R+ cells were maintained in Schneider’s Drosophila medium

(Invitrogen), with 10% heat inactivated fetal bovine serum

(Invitrogen), and 1% penicillin/streptomycin (Sigma-Aldrich).

For the primary screen, cells were spun down and resuspended

in serum-free medium at a density of 96104 cells/ml. 10 ml of the

cell suspension was added to each of the 384 wells using the Matrix

Wellmate 8-channel microplate dispenser (ThermoScientific;

DRSC). Cells were incubated for 30 min, then 30 ml of serum

containing medium were added to each well. Cells were cultured

for 5 d before the swell assay was performed (5 d RNAi treatment

is necessary to sufficiently knock down proteins with slow turnover

rates). For the secondary screen, cells were plated at a density of

40% in a 6 well dish. Once cells were adherent, the medium was

replaced with 1 ml of serum-free medium containing 0.015 mg/ml

dsRNA. Cells were incubated for 30 min at room temperature

followed by addition of 3 ml of serum-containing medium to each

well. Transfections were performed in duplicate; 1 well was used

for functional studies and the other for qPCR analysis of

knockdown at day 5.

Screening SolutionsIso-osmotic (320 milliOsm/kg; mOSM) solutions contained in

mM: 105 NaCl or NaI, 2 CaCl2, 1 MgCl2, 5 KCl, 10 HEPES, 10

Glucose, 90 Mannitol (pH 7.4 NaOH). For hypo-osmotic (240

mOSM) solutions, mannitol was omitted. 1 mM furosemide

(Sigma-Aldrich) blocked 100% of the S2R+ IClswell and prevented

significant H148Q-YFP suppression. Furosemide is a specific

blocker of Na-K-2Cl co-transporters (SLC12A2) at concentrations

in the mM range.

The Primary ScreenOur genome-wide screen was conducted at the Harvard/

HHMI Drosophila RNAi Screening Center using the DRSC 2.0

Genomewide RNAi Library. DRSC 2.0 is a collection of dsRNAs

for genome-wide RNAi knockdown covering , 13,900 genes

encoding proteins and non-coding RNAs while minimizing off-

target effects due to sequence similarity to other genes. Each gene

is targeted by 1.3 dsRNA/gene. The screen consisted of 66 384-

well assay plates in duplicate. Each well of the 384-well plate

contained 5 ml of 0.05 mg/ml dsRNA in water (0.25 mg dsRNA/

well). Each plate contained control RNAi specific for Thread

(Drosophila inhibitor of apoptosis protein), Rho (a small GTPase

activator of the EGFR signaling pathway), and GFP. On day 5 of

RNAi treatment, the cellular fluorescence of the H148Q-YFP

probe was measured under several treatment conditions using the

Analyst GT plate reader (Molecular Devices; DRSC). The probe

was excited at 485 nm and emissions collected at 530 nm. Before

fluorescence measurements were taken, the media was aspirated

(384-well aspirator; VP Scientific) and the cells were equilibrated

in 80 ml of 320 mOSM NaCl solution. After 10 min this solution

was removed by aspiration and cells incubated in 240 mOSM

NaCl for 5 min. Fluorescence was then measured, the NaCl

solution removed and cells were incubated in 240 mOSM NaI for

5 min. Fluorescence was again measured; the change in fluores-

cence was determined by dividing the fluorescence in 240 mOSM

NaI by that in 240 mOSM NaCl. Wells with fluorescence or ratio

changes (240 mOSM I2 fluorescence/240 mOSM Cl2 fluores-

cence) greater than 1.5 times the standard deviation (1.56S.D.) of

the plate mean were initially considered as hits (candidates of

Clswell channel or regulators of its activation pathway). False

positives could potentially result if the RNAi treatment caused a



high internal pH as H148Q-YFP has a pKa of 6.7. Cell death was

detected in control wells indicating effective RNAi treatment.

Generation of dsRNAcDNA templates were generated by PCR amplification of

genomic DNA using primers designed by the DRSC (SnapDragon

tool). These primers had the T7 promoter sequence (TAATAC-

GACTCACTATAGGG) added to the 5’ end of both primers.

The templates generally corresponded to exons but occasionally

sequences with two or more exons interrupted by introns were

used. The PCR fragments were ,150–600 base pairs in length,

and any complete 19-mer homology to other genes that could lead

to non-specific dsRNA are reported. Individual RNAi sequences

used here are found in the DRSC website (www.flyrnai.org).

dsRNAs against Drosophila were synthesized with the MEGAscript

in vitro transcription kit (Ambion). RNA was purified with the

RNeasyPlus mini kit (Qiagen) and stored at 280uC.

qPCR Analysis of RNAi EfficiencyAfter 5 d RNAi treatment, RNA was prepared from the S2R+

cells using the RNeasy Plus mini kit (Qiagen). 2.5 mg RNA was used

for each first-strand cDNA synthesis reaction (SuperScript Vilo

cDNA Synthesis kit, Invitrogen). Primers for qPCR were designed

on the NCBI/PrimerBlast site (http://www.ncbi.nlm. nih.gov/

tools/primer-blast/) with the following restrictions: PCR product

size was between 70 and 300 bp, primer melting temperatures were

between 57 and 63uC, primers spanned an exon-exon junction, and

primers were specific to the intended PCR template as determined

by BLAST analysis of the Drosophila melanogaster Refseq mRNA

database. Primer sets were only used if the melting curve had a

single peak. The RT2 Real-Time SYBR Green/Rox PCR master

mix (SABiosciences) was used for qPCR. qPCR reactions were set

up in quadruplicate to minimize pipetting errors, and run on the

Mastercycler ep Realplex real-time PCR system (Eppendorf).

Average cycle numbers for each primer set were normalized to

either dTaf8 or dAct79b average cycle numbers.

Secondary Screening of CandidatesComprehensive bioinformatics analysis of the hit list was

performed to identify potential candidates for Clswell. Hits were

limited to those with human homologs and at least a single

transmembrane domain. Potential regulators of the Clswell

activation pathway were left for future consideration. The effects

of RNAi on fluorescence changes were confirmed by plate reader

or imaging experiments. The specificity and effectiveness of the

RNAi was assessed by qPCR. S2R+ cells treated with RNAi were

patch clamped and IClswell was directly measured. Candidates were

cloned and expressed in HEK293 or CHO-K1 cells. IClswell was

measured by whole-cell patch clamp recording.

ElectrophysiologyWhole-cell patch clamp recordings were made at room

temperature. Recordings were obtained using an Axopatch

200B amplifier, Digidata 1322A analog-to-digital converter, and

pClamp 8.01 software (Molecular Devices, Union City, CA).

Data were low-pass filtered at 2 kHz and digitized at 5 kHz.

Fire-polished thin or thick wall borosilicate glass pipettes of 3–4

MV resistances were used for recordings; access resistance was

compensated to .80%. Cells were held at 270 mV to clearly

eliminate cells with leaky seals and voltage ramps (400 ms in

duration) from 2100 to +100 mV were applied every 2–5 s.

Liquid junction potentials were corrected during analysis, and

ramp data were plotted between –100 and +80 mV.

Recording SolutionsInternal pipette solution contained (in mM): 160 CsASP, 10

Cs4BAPTA, 4 MgATP, 2 MgCl2, 8 NaCl, and 10 HEPES (pH 7.4

with CsOH). 10 mM BAPTA was used to prevent activation of

channels by calcium and to reduce the endogenous HEK cell IClswell,

which is optimally activated with 100 nM Cai [47]. 240 mOSM

solution composition is detailed in ‘Screening solutions’. 200 mOSM

solutions contained in mM: 82.5 NaCl, NaI, NaSCN, NaMES, or

NaASP, 2 CaCl2, 1 MgCl2, 5 KCl, 10 HEPES, and 10 Glucose

(pH 7.4 with NaOH). 90 mM mannitol was added to bring

osmolality to 320mOSM.Therelativepermeabilities were estimated

fromtheGoldman-Hodgkin-Katzequation.Forourcalculations, the

[Cl]i was set to 0 mM. Cells were held at -70 mV during all

recordings, rapidly depleting Cli. Cation permeability was essentially

nil, as replacement of 200 mOSM NaCl solution with 200 mOSM

NMDG-Cl solution did not change the reversal potential (Erev; data

not shown). Slope conductances were calculated for each anionic

substitution between the Erev and +80 mV. For the S2R+ and HEK

cells Erev and I+80 mV were measured in 200 mOSM solutions. For

exogenously expressed dBest1, mBest2, and d64m Erev and I+80 mV

were measured in 320 mOSM solutions to prevent contamination

with the endogenous HEK cell IClswell. The HEK cell line chosen for

over-expression studies had no constitutive ICl (I320 mOSM did not

change when switching between Cl and ASP), the endogenous IClswell

developed very slowly, and the cells did not have an endogenous ISCN

(attributable to SLC1A family member expression [40].

PharmacologyStock solutions of DIDS (0.1 M in DMSO; Sigma), DCPIB

(50 mM in EtOH; Tocris), and furosemide (1M in DMSO; Sigma)

were prepared and diluted in 320 mOSM or 200 mOSM NaCl

solution to their final concentrations.

Molecular BiologydBest1 was a kind gift from Dr. Criss Hartzell (Emory University).

All other constructs were either ordered from Open Biosystems or

cloned from a Drosophila cDNA library or Human Brain (whole

Marathon ready cDNA library; BD Biosciences). Candidate cDNAs

were subcloned into pEGFP-N3 (C-terminal tag; BD Biosciences)

and an engineered Red pTracer vector (untagged). We found that an

N-terminal EGFP tag rendered dBest1 nonfunctional (data not

shown). Using site-directed mutagenesis we introduced the W94C

mutation intodBest1pEGFP-N3 (GeneArt site-directed mutagenesis,

Invitrogen, CA). dBest1 W94C-gfp was then subcloned into the

pAc5.1 V5-HisA vector (Invitrogen, CA).

Supporting Information

Figure S1 The late activating component of S2R+ IClswell

remains despite overexpression of dBest1 W94C-gfp. (A)

The late activating component of S2R+ IClswell is isolated after

dominant negative elimination of IdBest1. Inset: Step protocol. (B)

The late activating component of S2R+ IClswell is sharply rectifying

(ramp protocol; inset). Red trace is 320 mOSM solution, blue trace

is 80 s after the 200 mOSM solution change. The late activating

IClswell develops after 36 s in 200 mOSM solution.

(TIF)

Table S1 Secondary screening identifies Best1 as theDrosophila Clswell channel. Candidates with transmembrane

domains and human homologs were further studied to determine

if they formed the Clswell channel. 3 indicates a positive secondary

screening result; X indicates a negative result. B indicates that

several qPCR primer sets consistently had more than 1 melting



point peak suggesting nonspecific primer binding. The effective-

ness of RNAi knockdown, therefore, could not be determined by

qPCR. ?? indicates that two cells overexpressing SLC1A2 had

substantial ISCN- currents but small IClswell. Thus, SLC1A2

overexpression may upregulate endogenous HEK cell IClswell in

the majority of the population but does not form the channel itself.

HeLa cells treated with SLC1A3 siRNA (which reduced SLC1A2

and SLC1A3 mRNA by 90% and 92% respectively) had unaltered

IClswell (data not shown).

(TIF)

Acknowledgments

We thank the Drosophila RNAi Screening Center at Harvard Medical

School (NIH/NIGMS 2R01GM067761) for providing RNAi libraries,

laboratory space, bioinformatics tools and other support for the screen.

Author Contributions

Conceived and designed the experiments: SCS. Performed the exper-

iments: SCS. Analyzed the data: SCS. Contributed reagents/materials/

analysis tools: SCS DEC. Wrote the paper: SCS DEC.

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